Isolation of novel aav&#39;s and uses thereof

ABSTRACT

The invention in some aspects relates to isolated nucleic acids, compositions, and kits useful for identifying adeno-associated viruses in cells. In some aspects, the invention provides kits and methods for producing somatic transgenic animal models using recombinant AAV (rAAV) to an animal having at least one transgene that expresses a small interfering nucleic acid or at least one binding site for a miRNA.

RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. §120 of U.S.application Ser. No. 14/940,574, filed Nov. 13, 2015, entitled“Isolation of Novel AAV's and Uses Thereof”, which is a continuationunder 35 U.S.C. §120 of U.S. application Ser. No. 12/686,097, filed onJan. 12, 2010, entitled “Isolation of Novel AAV'S and Uses Thereof”,which is a continuation-in-part of U.S. Non-Provisional patentapplication Ser. No. 12/473,917, filed on May 28, 2009, entitled“Isolation of Novel AAV's and Uses Thereof”, which claims priority under35 U.S.C. §119(e) to U.S. provisional application, U.S. Ser. No.61/130,105, filed May 28, 2008, the entire contents of each applicationare incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersHL059407 and DK047757 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention in some aspects relates to isolated nucleic acids,compositions, and kits useful for identifying adeno-associated virusesin cells. In some aspects, the invention provides methods foridentifying adeno-associated viruses in cells based on the detection ofAAV RNA in the cells. In some aspects the invention provides methods forproducing somatic transgenic animal models using recombinant AAV withtissue targeting capabilities.

BACKGROUND OF INVENTION

Adeno associated Virus (AAV) is a small and helper dependent virus. Itwas discovered in 1960s as a contaminant in adenovirus (a cold causingvirus) preparations. Its growth in cells is dependent on the presence ofadenovirus and, therefore, it was named as adeno-associated virus.Before 2002, a total of 6 serotypes of AAVs were identified, includingthe serotype 2 which was the first AAV developed as vector for genetransfer applications and the one used in the recent break through eyegene therapy trials. In the earlier attempts to develop AAV as genetransfer vehicle, prototype AAV vector based on serotype 2 effectivelyserved as a proof-of-concept showcase and accomplished non-toxic andstable gene transfer in murine and large animal models in differenttarget tissues. For instance an 8 year stable vision improvement wasobserved in a dog model of LCA after a single injection and a 9 yearstable gene expression in Macaque muscle was achieved. However, theseproof-of-concept studies also revealed a significant shortcoming whichis a poor gene transfer efficiency in major target tissues.

Methods for discovering novel AAVs have been largely focused onisolating DNA sequences for AAV capsids, which relate to the tissuetargeting capacity of the virus. To date, the principal methods employedfor identifying novel AAV take advantage of the latency of AAV proviralDNA genomes and focus on rescuing persisted viral genomic DNA. The majorchallenge in DNA-targeted AAV isolation is that the abundance ofpersisted AAV genomes is often very low in most of tissues particularlyin human tissues, which makes AAV rescue unachievable in many cases.

SUMMARY OF INVENTION

In some aspects, the invention relates to the use of AAV based vectorsas vehicles for the development somatic transgenic animal models. Insome aspects, the invention relates to AAV serotypes that havedemonstrated distinct tissue/cell type tropism and can achieve stablesomatic gene transfer in animal tissues at levels similar to those ofadenoviral vectors (e.g., up to 100% in vivo tissue transductiondepending upon target tissue and vector dose) in the absence of vectorrelated toxicology. In other aspects, the invention relates to AAVserotypes having liver, heart and pancreas tissue targetingcapabilities. These tissues are associated with a broad spectrum ofhuman diseases including a variety of metabolic, cardiovascular anddiabetic diseases. Thus, in some aspects the invention relates to theuse of adeno-associated virus based vector as a vehicle for thedevelopment somatic transgenic animal models of human diseases such asmetabolic, cardiovascular, and diabetic disease.

Availability of appropriate animal models for further understandingpathogenesis and developing therapeutics for those diseases is a majorchallenge in biomedical research. The methods for generating the somaticmodels, as described herein, avoid the time consuming and costly processof germ line gene transfer and cogenic breeding for each target gene oftransgenic animals as well as some embryonic lethal consequences, andprovide versatility to create animal models from, for example, differentstrains, genetic background, or at different ages. The somatic animalmodels of the invention provide an important system for studyingpathogenesis of diseases and consequences of abnormal gene expression invarious tissue.

In other aspects the invention relates to methods involvingadministering a recombinant Adeno-Associated Virus (rAAV) to a subject,wherein the rAAV infects cells of a target tissue of the subject, andwherein the rAAV is at least one transgene that expresses a smallinterfering nucleic acid. The subject may be, for instance, an animalsuch as a somatic transgenic animal model.

In some embodiments the small interfering nucleic acid is an miRNA. Thesmall interfering nucleic acid may be an miRNA sponge that inhibits theactivity of at least one miRNA in the animal. For instance, the miRNA isan endogenous miRNA. In some embodiments the miRNA is expressed in acell of the target tissue, optionally wherein the target tissue isheart, liver, or pancreas. In other embodiments the transgene expressesa transcript that comprises at least one binding site for a miRNA,wherein the miRNA inhibits activity of the transgene, in a tissue otherthan the target tissue, by hybridizing to the binding site.

In some aspects a method is provided that involves administering arecombinant Adeno-Associated Virus (rAAV) to a subject, wherein the rAAVcomprises at least one transgene, wherein the transgene expresses atranscript that comprises at least one binding site for a miRNA, whereinthe miRNA inhibits activity of the transgene, in a tissue other than atarget tissue, by hybridizing to the binding site of the transcript.

In other aspects, a method for generating a somatic transgenic animalmodel by administering a recombinant Adeno-Associated Virus (rAAV) to ananimal, wherein the rAAV comprises at least one transgene, wherein thetransgene expresses a transcript that comprises at least one bindingsite for a miRNA, wherein the miRNA inhibits activity of the transgene,in a tissue other than a target tissue, by hybridizing to the bindingsite of the transcript is provided.

In some embodiments of the methods provided herein the transgene is atissue specific promoter or inducible promoter. The tissue specificpromoter may be, for instance, a liver-specific thyroxin bindingglobulin (TBG) promoter, a insulin promoter, a glucagon promoter, asomatostatin promoter, a pancreatic polypeptide (PPY) promoter, asynapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammaliandesmin (DES) promoter, a α-myosin heavy chain (a-MHC) promoter, or acardiac Troponin T (cTnT) promoter.

In some embodiments the rAAV has a capsid from an AAV serotype selectedfrom: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, and AAV12. In other embodiments the rAAV is a variant of an AAVserotype selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, and AAV12. In yet other embodiments the rAAV has anInverted Terminal Repeat (ITR) sequence from an AAV serotype selectedfrom: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, and AAV12.

The target tissue may be any tissue such as gonad, diaphragm, heart,stomach, liver, spleen, pancreas, or kidney. The rAAV may transduces anytype of tissue, for example, muscle fibers, squamous epithelial cells,renal proximal or distal convoluted tubular cells, mucosa gland cells,blood vessel endothelial cells, or smooth muscle cells.

In some embodiments a dose of 10¹⁰, 10¹¹, 10¹², or 10¹³ genome copies isadministered.

In other embodiments the methods of administering are performedintravenously, or through the portal vein of the animal.

In some embodiments the subject is a human or an animal, wherein theanimal is a mammal, optionally wherein the mammal is selected from amouse, a rat, a rabbit, a dog, a cat, a sheep, a pig, a non-humanprimate.

The transgene may express, for instance, a cancer related gene, anapoptosis-related gene, or a pro-apoptotic gene.

In some embodiments the transgene causes a pathological state in theanimal.

The method may also include the step of administering a putativetherapeutic agent to the somatic transgenic animal model to determinethe effect of the putative therapeutic agent on the pathological statein the animal.

In some embodiments the transgene expresses a reporter gene. Thereporter gene may be a reporter enzyme, optionally which isBeta-Galactosidase or a Fluorescent protein, optionally which is GFP.

In some embodiments the transgene expresses a transcript that comprisesbinding sites for different microRNAs that are endogenously expressed indifferent cells of the animal and that inhibit activity of the transgenein different tissues.

A somatic transgenic animal produced by the method described herein isalso provided according to the invention.

A kit for producing a rAAV that generates a somatic transgenic animalthat expresses a transgene in a target tissue is provided according toother aspects of the invention. The kit includes at least one containerhousing a recombinant AAV vector, wherein the recombinant AAV vectorcomprises a transgene that expresses a small interfering nucleic acidand/or expresses a transcript that comprises at least one binding sitefor a miRNA, wherein the miRNA inhibits activity of the transgene, in atissue other than the target tissue, by hybridizing to the binding siteof the transcript, at least one container housing a rAAV packagingcomponent, and instructions for constructing and packaging the rAAV,wherein the rAAV transduces cells of the target tissue.

In some embodiments a rAAV packaging component includes a host cellexpressing at least one rep gene and/or at least one cap gene.

In other embodiments the host cell is a 293 cell.

The host cell, in some embodiments, expresses at least one helper virusgene product that effects the production of rAAV containing therecombinant AAV vector. The at least one cap gene may encode a capsidprotein from an AAV serotype that binds to cells of the target tissue.

In some embodiments the AAV serotype is selected from: AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In otherembodiments the AAV serotype is a variant of an AAV serotype is selectedfrom: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, and AAV12. In yet other embodiments the rAAV has an InvertedTerminal Repeat (ITR) sequence from an AAV serotype selected from: AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

In some embodiments a rAAV packaging component includes a helper virusoptionally wherein the helper virus is an adenovirus or a herpes virus.

The target tissue may be any tissue such as gonad, diaphragm, heart,stomach, liver, spleen, pancreas, or kidney. The rAAV may transduces anytype of tissue, for example, muscle fibers, squamous epithelial cells,renal proximal or distal convoluted tubular cells, mucosa gland cells,blood vessel endothelial cells, or smooth muscle cells.

The transgene may express, for instance, a cancer related gene, anapoptosis-related gene, or a pro-apoptotic gene.

In some embodiments the small interfering nucleic acid is an miRNA. Thesmall interfering nucleic acid may be an miRNA sponge, wherein miRNAsponge inhibits the activity of one or more miRNAs in the somatictransgenic animal.

In other embodiments the miRNA is an endogenous miRNA of the animal. ThemiRNA may be expressed in a cell of a heart, liver, or pancreas tissuein the somatic transgenic animal.

In some embodiments the transgene expresses a toxin and/or a reportergene. The reporter gene may be a reporter enzyme, optionally which isBeta-Galactosidase or a Fluorescent protein, optionally which is GFP.

In some embodiments the transgene includes a tissue specific promoter orinducible promoter. The tissue specific promoter may be a liver-specificthyroxin binding globulin (TBG) promoter, a insulin promoter, a glucagonpromoter, a somatostatin promoter, a pancreatic polypeptide (PPY)promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter,a mammalian desmin (DES) promoter, a α-myosin heavy chain (a-MHC)promoter, or a cardiac Troponin T (cTnT) promoter.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIGS. 1A-1B depict the results of an assessment of the ability of rAAVsto deliver transgenes that are susceptible to miRNA targeting anddegradation. FIG. 1A depicts multiple AAV expression constructs.Schematics of transcript length and cleavage products are shown next tobands which correspond to AAV+miRBS. FIG. 1B shows that expression ofAAV−miRBS, which contains binding sites for miR-122 does notsignificantly affect miR-122 expression compared with wild-type control,or mRNA or protein expression of the miR-122 target gene, Cyclin G1.

FIG. 2A is a schematic of a recombinant AAV, rAAV9.CB.nLacZ.3xmiR122,that contains a LacZ coding sequence flanked by 5′ and 3′ invertedterminal repeat sequences. rAAV.CB.nLacZ.3xmi122 also has a ubiquitousEM7 promoter, CMV immediate early enhancer and chicken beta-actinpromoter sequences, and three miR-122 binding sites in the 3′ UTR. FIG.2B shows that mice infected with rAAV.CB.nLacZ.3xmiR122 exhibitdetectable LacZ expression (as detected by X-Gal staining) in hearttissue but not liver tissue, where miR-122 is expressed, whereas acontrol rAAV that does not contain miR122 binding sites is expressed inboth heart and liver tissue.

FIG. 3 depicts X-Gal staining of heart, pancreas and liver tissuesections from mice infected with recombinant AAVs that express a LacZreporter gene controlled by a chicken beta actin promoter, each havingmiR-122 binding sites and/or miR-1 binding sites in its 3′ UTR. ControlrAAVs, which express a LacZ reporter gene controlled by a chicken betaactin promoter without miRNA binding sites, produced high levels of LacZexpression in heart and liver tissue. (first row.) rAAVs with a singlemiR-122 site in the 3′-UTR produced high levels of LacZ reporter geneexpression in heart tissue but not pancreas or liver tissue. (secondrow.) rAAVs with a single (or three) miR-1 site(s) in the 3′-UTRproduced high levels of LacZ reporter gene expression in liver tissuebut not pancreas or heart tissue. (third and fourth rows.) rAAVs with asingle miR-1 site and a single miR-122 site in the 3′-UTR did notproduce significant levels of LacZ reporter gene expression in any ofthe three tissues. (fifth row.)

FIG. 4 depicts rAAV-9-based Let-7 sponge delivery vector having 5′- and3′-inverted terminal repeat regions, a CMV immediate early enhancer, achicken beta-actin promoter, a luciferase reporter gene, one or moresponge sequences, and a globin poly-A tail sequence.

FIG. 5A depicts live luciferase imaging of mice four weeks after IVadministration of either rAAV9CB.FFLuc.7XLet7 sponge or rAAV9CB.FFLuc.7XLet7-mutant sponge. FIG. 5B shows that luciferase expression(photons/sec) was persistent up to four weeks post injection.

FIG. 6A shows a comparison of luciferase radiance (photons/sec) betweenlet-7 and mutant let-7 treated mice at the same injection dose fromwhole body imaging. FIG. 6B shows a comparison of luciferasefluorescence density (RLU/ug protein) between let-7 and mutant let-7treated mice at the same injection dose from liver homogenates. FIG. 6Cshows that transgene copy numbers per cell in animal livers weredependent on injection dose (low dose (LD), 1E11 genomic copies/mouse orhigh dose (HD), 1E12 genomic copies/mouse).

FIG. 7 shows the structure of miR122 sponge sequences, which whereincorporated into rAAV-9-based miR-122 sponge delivery vectors. SEQ IDNO: 13 corresponds to miR-133; SEQ ID NO: 14 corresponds to miR-122sponge; SEQ ID NO: 15 corresponds to MutmiR-122 sponge; and SEQ ID NO:16 corresponds to 7xmiR-122 sponge.

FIG. 8 depicts live luciferase imaging of C57BL/6 mice three weeks afterIV administration of either rAAV9TBG.FFLuc.7X miR-122 sponge or rAAV9rAAV9TBG.FFLuc.7X miR-122-mutant sponge. In both cases luciferaseactivity was detected mainly in the abdomen.

FIG. 9 depicts a bar graph of a comparison of luciferase expression, atmultiple time points, between mice injected with a low dose (1E11genomic copies/mouse) or a high dose (1E12 genomic copies/mouse) ofrAAV9CB.FFLuc.7X miR-122 sponge.

FIG. 10A depicts a bar graph of luciferase expression at multiple timepoints in mice injected with rAAV9CB.FFLuc.7X miR-122-mutant sponge(1E12 genomic copies/mouse). FIG. 10B depicts a bar graph of acomparison of luciferase expression, at multiple time points, betweenmice injected with rAAV9CB.FFLuc.7X miR-122 sponge or rAAV9CB.FFLuc.7XmiR-122-mutant sponge (1E12 genomic copies/mouse).

FIGS. 11A-11D depict primer design and PCR analysis in mice of lacZmRNA. FIG. 11A depicts three primer set locations for amplifying LacZmRNAs. FIG. 11B depicts hypothetical RT-PCR results with both oligo dTand random primer RT steps. FIG. 11C depicts RT-PCR results using primerset 3 of FIG. 11A. FIG. 11D depicts RT-PCR results using primer sets 1and 2, with oligo dT or random priming RT steps. For FIGS. 11C and 11D,RNA was obtained from mice expressing LacZ without miRNA binding sites(AAV) or with miRNA binding sites (AAV+miRBS).

FIG. 12 depicts northern blot analysis of LacZ mRNA cleavage.

FIG. 13 depicts MiR-122 specific rescue and sponge target validation.

FIG. 14 depicts a Let-7 sponge design. SEQ ID NO: 16 corresponds toLet-7b; SEQ ID NO: 17 corresponds to Let-7b sponge; SEQ ID NO: 18corresponds to MutLet-7b sponge; and SEQ ID NO: 19 corresponds to7xLet-7b sponge.

DETAILED DESCRIPTION

Adeno-associated virus (AAV) is a small (20 nm) replication-defective,nonenveloped virus, that depends on the presence of a second virus, suchas adenovirus or herpes virus, for its growth in cells. AAV is not knownto cause disease and induces a very mild immune response. AAV can infectboth dividing and non-dividing cells and may incorporate its genome intothat of the host cell. These features make AAV a very attractivecandidate for creating viral vectors for gene therapy. Prototypical AAVvectors based on serotype 2 provided a proof-of-concept for non-toxicand stable gene transfer in murine and large animal models, butexhibited poor gene transfer efficiency in many major target tissues.The invention in some aspects seeks to overcome this shortcoming byproviding methods for identifying novel AAVs having distinct tissuetargeting capabilities for gene therapy and research applications.

The biology of AAV vector is primarily dictated by its capsid.Consequently, methods for discovering novel AAVs have been largelyfocused on isolating DNA sequences for AAV capsids. To date, the primarymethods used for isolating novel AAV include PCR based molecular rescueof latent AAV DNA genomes, infectious virus rescue of latent proviralgenome from tissue DNAs in vitro in the presence of adenovirus helperfunction, and rescue of circular proviral genome from tissue DNAs byrolling-circle-linear amplification, mediated by an isothermal phagePhi-29 polymerase. All of these isolation methods take advantages of thelatency of AAV proviral DNA genomes and focus on rescuing persistedviral genomic DNA. The major challenge in DNA-targeted AAV isolation isthat the abundance of persisted AAV genomes is often very low in mosttissues particularly in human tissues, which makes AAV rescueunachievable in many cases.

The invention in some aspects is based on the surprising discovery thatendogenous latent AAV genomes are transcriptionally active in mammaliancells (e.g., cells of nonhuman primate tissues such as liver, spleen andlymph nodes). A central feature of the adeno-associated virus (AAV)latent life cycle is persistence in the form of integrated and/orepisomal genomes in a host cell. However, prior to the instant inventionit was not known whether AAVs express viral genes (e.g., rep and capgenes) during latency (i.e., in a latent state).

As disclosed herein, both rep and cap gene transcripts are detected withvariable abundances by RNA detection methods (e.g., RT-PCR). Presence ofcap gene transcripts and ability to generate cDNA of cap RNA throughreverse transcription (RT) in vitro significantly increase abundance oftemplates for PCR-based rescue of novel cap sequences from tissues andenhance the sensitivity of novel AAV recovery. In some aspects, themethods of the invention involve transfecting cells with total cellularDNAs isolated from the tissues that potentially harbor proviral AAVgenomes at very low abundance and supplementing with helper virusfunction (e.g., adenovirus) to trigger and/or boost AAV rep and cap genetranscription in the transfected cells. In some cases, RNA from thetransfected cells provides a template for RT-PCR amplification of cDNA,for example, cap cDNA.

Thus, the invention provides methods for detecting an adeno-associatedvirus (AAV) in a cell based on the detection of an RNA in the cell thatis indicative of the presence of the adeno-associated virus in the cell.The cell may be obtained from any number of sources such as from atissue, saliva, gingival secretions, cerebrospinal fluid,gastrointestinal fluid, mucus, urogenital secretions, synovial fluid,blood, serum, plasma, urine, cystic fluid, lymph fluid, ascites, pleuraleffusion, interstitial fluid, intracellular fluid, ocular fluids,seminal fluid, mammary secretions, vitreal fluid, and nasal secretions.In some specific cases, the cell is from liver, spleen, or lymph nodetissue. The cells may be obtained from any number of sources usingmethods well known in the art.

RNA may be isolated from a cell (preferably a cell known to have alatent AAV infection) and using any number of appropriate methods todetect the RNA, such as those described below. For the purposes ofidentifying novel AAVs it is useful to employ any RNA detection methodknown in the art that produces sequence information about the RNA. Thesequence information can then be used to identify the AAV.

Typically, the isolated RNA is reverse transcribed to produce cDNA. ThecDNA can then be easily manipulated to obtain sequence information aboutthe template RNA. For example, in some cases the cDNA can be cloned intoany appropriate vector plasmid (e.g., cloning plasmid) and amplified(e.g., in bacteria). The cloned cDNA can then be purified and sequencedusing methods well known in the art.

Still other RNA detection methods are useful in the current invention.For example, the invader assay has been developed to detect RNAmolecules, using a two-step FRET-based invader assay (Kwiatkowski, R.W., Lyamichev, V., de Arruda, M. and Neri, B. (1999) Clinical, genetic,and pharmacogenetic applications of the Invader assay. Mol Diagn, 4(4),353-64.). High-throughput parallel analyses of many RNA transcripts arepossible with DNA microarray technology. Padlock probes are useful todetect single-nucleotide variants in RNA. Serial analysis of geneexpression (SAGE) is another approach. Other approaches will be apparentto the skilled artisan.

In some embodiments, isolated RNA from a cell having a latent AAV isreverse transcribed to produce cDNA. The cDNA is then subjected to a PCRreaction to amplify a target sequence of interest. Any number of PCRmethods known in the art may be appropriate including, for example,Allele specific PCR, Asymmetric PCR, Solid Phase PCR, TAIL-PCR: Thermalasymmetric interlaced PCR, Hot-start PCR, Touchdown PCR, Inverse PCR,Linear PCR, Ligation-mediated PCR, Multiplex-PCR, Nested PCR, andQuantitative PCR (Q-PCR) (e.g., Taqman, SYBR green).

Typically, the target sequence of the PCR has useful information aboutthe AAV serotype. The target sequence may span about 100 bp to about 2.8kilobase pairs in length. It is particularly desirable that the targetsequence is sufficiently unique to positively identify the amplifiedsequence as being from a particular AAV serotype. For example, in oneembodiment, the target sequence is about 250 bp in length, and issufficiently unique among known AAV sequences, that it positivelyidentifies the amplified region as being of AAV unique origin. Typicallythe target sequence will contain a variable sequence (e.g., AAVhypervariable region) that is sufficiently unique that can be used toidentify the AAV serotype from which the amplified sequences originate.Once amplified (and thereby detected), the sequences can be identifiedby performing conventional restriction digestion and comparison torestriction digestion patterns for this region in any of AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or anyother known or novel serotypes. The skilled artisan will appreciate thatthe sequences of known serotypes can be obtained from various publiclyaccessible databases including for example GenBank. In preferredembodiments, the amplified (and thereby detected) target sequences areidentified by sequencing and comparing the obtained sequence to knownAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12 serotypes, or any other novel serotypes.

The invention, thus, includes isolated nucleic acids useful foridentifying novel AAV sequences. Typically, the isolated nucleic acidsare used as primers in reverse transcription and/or polymerase chainreactions. Examples of primers useful in a reverse transcriptionreaction include OligodT, random hexamers, and the sequence specificprimers disclosed herein (e.g., SEQ ID NO 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4). Other primers appropriate for a reverse transcriptionreaction are known in the art. Nucleic acids of the invention which areuseful as PCR primers may have a sequence that has substantial homologywith a nucleic acid sequence of a region that is highly conservedbetween at least two AAV serotypes. In some cases, the region is highlyconserved between two, three, four, five, six, seven, eight, nine, ten,eleven, or twelve or more AAV serotypes. Typically, the region that ishighly conserved covers an end-to-end length of between 25 and 250 bp.In specific cases, the region covers about 150 bp. However, in othercases the end-to-end length of the highly conserved region is greaterthat 250 bp. Preferably, the region is highly conserved within thisend-to-end length over at least about 9, and more preferably, at least18 base pairs (bp). However, the region may be conserved over more than18 bp, more than 25 bp, more than 30 bp, or more than 50 bp.

The term “substantial homology”, when referring to a nucleic acid, orfragment thereof, indicates that, when optimally aligned withappropriate nucleotide insertions or deletions with another nucleic acid(or its complementary strand), there is nucleotide sequence identity inabout 90 to 100% of the aligned sequences.

The term “highly conserved” means at least 80% identity, preferably atleast 90% identity, and more preferably, over 97% identity. In somecases, highly conserved may refer to 100% identity. Identity is readilydetermined by one of skill in the art by, for example, the use ofalgorithms and computer programs known by those of skill in the art.

As described herein, alignments are performed using any of a variety ofpublicly or commercially available Multiple Sequence Alignment Programs,such as “Clustal W”, accessible through Web Servers on the internet.Alternatively, Vector NTI utilities are also used. There are also anumber of algorithms known in the art which can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta, a program in GCG Version 6.1. Fasta providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta with its default parameters (a word size of 6 and the NOPAM factorfor the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Similar programs are available for amino acidsequences, e.g., the “Clustal X” program. Generally, any of theseprograms are used at default settings, although one of skill in the artcan alter these settings as needed. Alternatively, one of skill in theart can utilize another algorithm or computer program which provides atleast the level of identity or alignment as that provided by thereferenced algorithms and programs.

Typically, the AAV serotypes (e.g., at least two) are selected fromAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, andAAV12. However, the invention is not so limited and other appropriateAAV serotypes (and subtypes) will be apparent to one of ordinary skillin the art.

In some cases, an isolated nucleic acid of the invention is a primerthat has substantial homology with a nucleic acid sequence correspondingto a 5′ or 3′ untranslated region of a transcript (e.g., mRNA) of anAAV. AAV genes include the cap proteins, including the VP1, VP2, VP3,and the rep proteins, including Rep78, Rep68, Rep52, and Rep40. Theoverlapping sequences of three capsid proteins, VP1, VP2 and VP3, aregenerally understood to be transcribed from one promoter, designatedp40. Whereas, two promoters identified as p5 and p19, are understood toproduce transcripts encoding Rep78, Rep68, Rep52 and Rep40. Thus, insome cases, an isolated nucleic acid of the invention (e.g., primer)comprises a nucleic acid sequence that has substantial homology with anucleic acid sequence corresponding to a 5′ or 3′ untranslated region ofa p5, p19, or p40 initiated transcript (i.e., mRNA) of an AAV. Theprimer may be, for instance, a nucleic acid sequence that hassubstantial homology with a nucleic acid sequence corresponding to a 5′untranslated region of a p40 initiated transcript (i.e., Cap gene mRNA)of an AAV. Alternatively, the primer may be a nucleic acid sequence thathas substantial homology with a nucleic acid sequence corresponding to a3′ untranslated region of a p40 initiated transcript (i.e., Cap genemRNA) of an AAV. In some embodiments the primers have a sequence as setforth in Table 1.

TABLE 1 AAV CAP GENE PRIMERS SEQ ID NUCLEIC ACID SEQUENCE SEQ ID NO: 1CapF-X(A/G/C/T/absent)GA(C/T)TG(C/T)(A/G/C)(A/T)(C/T/A)(A/T)(C/T)(G/T)GA(A/G)CAATAAATGA(A/G/C/T/absent) SEQ ID NO: 1- CapF-XNGAYTGYVWHWYKGARCAATAAATGAN (single letter code) SEQ ID NO: 2 CapR-X(A/G/C/T/absent)GAAACGAAT(C/A/T)AA(C/A)CGGTTTATTGATTAA(A/ G/C/T/absent)SEQ ID NO: 2- CapR-X NGAAACGAATHAAMCGGTTTATTGATTAAN (single letter code)SEQ ID NO: 3 CapF GACTGTGTTTCTGAGCAATAAATGA SEQ ID NO: 4 CapRGAAACGAATTAACCGGTTTATTGATTAA SEQ ID NO: 5 CapF22-X(A/G/C/T/absent)(C/T)(C/A)(A/G)(T/A)(C/A)(NG)(A/T)C(G/T)(G/T)(G/C)AGA(A/C)GCGG(A/G)(A/C)(G/C)(A/G/C/T/absent) SEQ ID NO: 5 CapF22-XNYMRWMRWCKKSAGAMGCGGRMSN (single letter code) SEQ ID NO: 6 CapF22CCATCGACGTCAGACGCGGAAG SEQ ID NO: 7 CapF64-X(A/G/C/T/absent)(G/C)(G/C)(C/A/G)GAC(A/G)(G/C)(G/C)T(A/C)(G/C)CA(A/G)(A/T)(A/T)CA(A/G)A(T/C)GT(A/G/C/T/absent) SEQ ID NO: 7 CapF64-XNSSVGACRSSTMSCARWWCARAYGTN (single letter code) SEQ ID NO: 8 CapF64GCCGACAGGTACCAAAACAAATGT SEQ ID NO: 9 CapF201-(A/G/C/T/absent)(C/A)(C/T)GG(C/A)(G/A)(T/C)GT(C/G)A(G/A)(A/T)AT X(C/T)T(C/G)AA(C/T)C(A/G/C/T/absent) SEQ ID NO: 9 CapF201-NMYGGMRYGTSARWATYTSAAYCN (single letter X code) SEQ ID NO: 10 CapF201CCGGCGTGTCAGAATCTCAACC SEQ ID NO: 11 AV2cas-X(A/G/C/T/absent)AC(A/G)(C/G/T)(A/G)AGANCCAAAGTTCAACTGA(A/C)ACGA(A/G/C/T/absent) SEQ ID NO: 11 AV2cas-XNACRBRAGANCCAAAGTTCAACTGAMACGAN (single letter code) SEQ ID NO: 12AV2cas ACAGGAGACCAAAGTTCAACTGAAACGA

In some embodiments, the PCR methods of the invention comprise a firstprimer having the sequence as set forth in SEQ ID NO: 1 and a secondprimer having a sequence as set forth in SEQ ID NO: 2. In someembodiments, the PCR methods of the invention comprise a first primerhaving the sequence as set forth in SEQ ID NO: 3 and a second primerhaving a sequence as set forth in SEQ ID NO: 4.

The target sequence obtained in the PCR reaction may be all or a portionof the cDNA In some cases the cDNA is about 50, about 100, about 250,about 500, about 1000, about 2000, about 4000 base pairs in length. Incertain cases, the cDNA is approximately 2300 base pairs, approximately2600 base pairs, or approximately 4700 base pairs in length. However,the invention is not so limited and the actual cDNA length will dependon a variety of factors such as AAV serotype, RT reaction primers, RTreaction condition. In most cases, the cDNA has a length that issufficient to obtain unique sequence information that can be used toidentify the AAV serotype from which the amplified sequences originate.

The target sequence obtained in the PCR reaction may be all or a portionof one or more AAV rep or cap genes, such as VP1, VP2 and VP3.Alternatively, the target sequence obtained in the PCR reaction may beall or a portion of one or more AAV hypervariable regions. In the caseswhere a portion of a gene (e.g., VP1, VP2, or VP3) is obtained it isunderstood that the portion will be of a sufficient size and from anappropriate position within the gene (e.g., coding region, variableregion) to provide unique sequence information that can be used toidentify the AAV serotype from which the amplified sequences originate.

The PCR primers are generated using techniques known to those of skillin the art. Each of the PCR primer sets is composed of a forward primer(i.e., 5′ primer) and a reverse primer (i.e., 3′ primer). See, e.g.,Sambrook J et al. 2000. Molecular Cloning: A Laboratory Manual (ThirdEdition). The term “primer” refers to an oligonucleotide which providesas a point of initiation of synthesis when placed under conditions (PCRreaction) in which synthesis of a primer extension product which iscomplementary to a nucleic acid strand is induced. The primer ispreferably single stranded. However, if a double stranded primer isutilized, it is treated to separate its strands before being used toprepare extension products. The primers may be about 15 to 30 or morenucleotides, and preferably at least 18 nucleotides. However, forcertain applications shorter nucleotides, e.g., 7 to 15 nucleotides areutilized. In certain embodiments, the primers are about 25 nucleotideslong (e.g., SEQ ID NO 3 or 4)

The primers are selected to be sufficiently complementary to thedifferent strands of each specific sequence to be amplified such thatthey hybridize with their respective strands. Typically, hybridizationoccurs under standard PCR conditions known in the art. Thus, primershaving melting temperatures between 50 and 65° C. are normally suitable.However, the invention is not so limited. In addition, the primersequence need not reflect the exact sequence of the region beingamplified. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer (e.g., for cloning purposes), withthe remainder of the primer sequence being substantially (e.g.,completely) complementary to the strand. In some cases, a primer mayinclude a sequence (e.g., 5′ sequence) that is not substantiallycomplementary to the target sequence but that facilitates subsequentmanipulation of the amplicon (e.g., cDNA). For example, in some cases, aprimer may have additional sequence at its 5′ end having a uniquerestriction site that facilitates subsequent digestion by an appropriaterestriction enzyme. Methods such as this can be employed to accomplish,for example, a cloning step. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thestrand to be amplified to hybridize therewith and form a template forsynthesis of the extension product of the other primer. Techniques suchas these and others disclosed herein are well known in the art and aresuitable for use in the methods of the instant invention.

The PCR primers for amplifying the target sequence according to theinvention are based upon the highly conserved sequences of two or morealigned sequences (e.g., two or more AAV serotypes). The primers canaccommodate less than exact identity among the two or more aligned AAVserotypes at the 5′ end or in the middle. However, the sequences at the3′ end of the primers correspond to a region of two or more aligned AAVserotypes in which there is exact identity over at least five,preferably, over at least nine base pairs, and more preferably, over atleast 18 base pairs at the 3′ end of the primers. Thus, the 3′ end ofthe primers is composed of sequences with 100% identity to the alignedsequences over at least five nucleotides. However, one can optionallyutilize one, two, or more degenerate nucleotides at the 3′ end of theprimer.

As disclosed herein, both rep and cap gene transcripts are detected withvariable abundances by RNA detection methods (e.g., RT-PCR). Theexpression of cap gene transcripts and ability to generate cDNA of capRNA through reverse transcription (RT) using the methods disclosedherein, significantly increase abundance of templates for PCR-basedrescue of novel cap sequences from tissues. The methods of the inventionin certain aspects, are useful for isolating novel full lengthfunctional cap cDNA sequences. The methods involve the design andselection of oligonucleotide primers for both RT and PCR reactions. Asdiscussed herein, AAV cap gene transcription is directed by AAV p40promoter which is located in the coding sequence of rep genes. Thus, insome cases, the region between the beginning of p40 RNA transcript andthe start codon of capsid VP1 cDNA is a target for the 5′ primers toretrieve the intact 5′ end of cap cDNA. In order to recover the intact3′ end of the cap transcript, the 3′ primer is typically selected in theregion of the polyadenylation signal. However, the invention is not solimited and other similar strategies can be employed to isolate novelcDNA sequences of this and other AAV genes.

In some cases, multiple primer sets can be used to isolate novel cDNAsequences of an AAV gene in fragments. Fragments so obtained can, forexample, be cloned together to form a single cDNA comprising a completegene sequence. For example, a first primer set having a 5′ primercomplementary to an untranslated region of an AAV gene and a 3′ primer(anchor primer) complementary to a sequence within the AAV transcript(e.g., in an intronic or exonic sequence) can be used to obtain a firstfragment (e.g., a 5′ fragment of a gene sequence). A second primer set,having a 5′ primer (e.g., anchor primer) complementary to a sequenceupstream of the second 3′ primer of the first primer set and a 3′ primercomplementary to a position near the polyadenylation signal can be usedto obtain a second fragment (e.g., a 3′ fragment of a gene sequence).The two fragments can have any number of uses thereafter, for examplethey can be analyzed separately (e.g., sequenced) or cloned together toobtain a complete gene sequence. In some cases, three, four, five, sixor more primer sets can be used to obtain three, four, five, six or moreof AAV gene fragments. Moreover, these examples are not meant to belimiting and any number of primer sets can be employed to obtain anynumber of fragments provided that the fragments are useful foridentifying and obtaining unique AAV sequences (e.g., Capsid genesequences).

In some cases, the methods involve transfecting cells with totalcellular DNAs isolated from the tissues that potentially harbor proviralAAV genomes at very low abundance and supplementing with helper virusfunction (e.g., adenovirus) to trigger and/or boost AAV rep and cap genetranscription in the transfected cell. In some cases, RNA from thetransfected cells provides a template for RT-PCR amplification of cDNAand the detection of novel AAVs. In cases where cells are transfectedwith total cellular DNAs isolated from the tissues that potentiallyharbor proviral AAV genomes, it is often desirable to supplement thecells with factors that promote AAV gene transcription. For example, thecells may also be infected with a helper virus, such as an Adenovirus ora Herpes Virus. In a specific embodiment, the helper functions areprovided by an adenovirus. The adenovirus may be a wild-type adenovirus,and may be of human or non-human origin, preferably non-human primate(NHP) origin. Similarly adenoviruses known to infect non-human animals(e.g., chimpanzees, mouse) may also be employed in the methods of theinvention (See, e.g., U.S. Pat. No. 6,083,716). In addition to wild-typeadenoviruses, recombinant viruses or non-viral vectors (e.g., plasmids,episomes, etc.) carrying the necessary helper functions may be utilized.Such recombinant viruses are known in the art and may be preparedaccording to published techniques. See, e.g., U.S. Pat. No. 5,871,982and U.S. Pat. No. 6,251,677, which describe a hybrid Ad/AAV virus. Avariety of adenovirus strains are available from the American TypeCulture Collection, Manassas, Va., or available by request from avariety of commercial and institutional sources. Further, the sequencesof many such strains are available from a variety of databasesincluding, e.g., PubMed and GenBank.

Cells may also be transfected with a vector (e.g., helper vector) whichprovides helper functions to the AAV. The vector providing helperfunctions may provide adenovirus functions, including, e.g., E1a, E1b,E2a, E4ORF6. The sequences of adenovirus gene providing these functionsmay be obtained from any known adenovirus serotype, such as serotypes 2,3, 4, 7, 12 and 40, and further including any of the presentlyidentified human types known in the art. Thus, in some embodiments, themethods involve transfecting the cell with a vector expressing one ormore genes necessary for AAV replication, AAV gene transcription, and/orAAV packaging.

In some cases, a novel isolated capsid gene can be used to construct andpackage recombinant AAV vectors, using methods well known in the art, todetermine functional characteristics associated with the novel capsidprotein encoded by the gene. For example, novel isolated capsid genescan be used to construct and package recombinant AAV (rAAV) vectorscomprising a reporter gene (e.g., B-Galactosidase, GFP, Luciferase,etc.). The rAAV vector can then be delivered to an animal (e.g., mouse)and the tissue targeting properties of the novel isolated capsid genecan be determined by examining the expression of the reporter gene invarious tissues (e.g., heart, liver, kidneys) of the animal. Othermethods for characterizing the novel isolated capsid genes are disclosedherein and still others are well known in the art.

The invention also involves the production of somatic transgenic animalmodels of disease using recombinant Adeno-Associated Virus (rAAV) basedmethods. A somatic transgenic animal model is a non-human animal. Themethods are based, at least in part, on the observation that AAVserotypes mediate efficient and stable gene transfer in a tissuespecific manner in adult animals. The rAAV elements (capsid, promoter,transgene products) are combined to achieve somatic transgenic animalmodels that express a stable transgene in a time and tissue specificmanner. The somatic transgenic animal produced by the methods of theinvention can serve as useful models of human disease, pathologicalstate, and/or to characterize the effects of gene for which the function(e.g., tissue specific, disease role) is unknown or not fullyunderstood. For example, an animal (e.g., mouse) can be infected at adistinct developmental stage (e.g., age) with a rAAV comprising a capsidhaving a specific tissue targeting capability (e.g., liver, heart,pancreas) and a transgene having a tissue specific promoter drivingexpression of a gene involved in disease. Upon infection, the rAAVinfects distinct cells of the target tissue and produces the product ofthe transgene.

In some embodiments, the transgene causes a pathological state. Atransgene that causes a pathological state is a gene whose product has arole in a disease or disorder (e.g., causes the disease or disorder,makes the animal susceptible to the disease or disorder) and/or mayinduce the disease or disorder in the animal. The animal can then beobserved to evaluate any number of aspects of the disease (e.g.,progression, response to treatment, etc). These examples are not meantto be limiting, other aspects and examples are disclosed herein anddescribed in more detail below.

The rAAVs useful in the methods of the invention may be those uniquerAAVs identified using the methods described herein or may be existingrAAVs known in the art. The rAAVs preferably have tissue-specifictargeting capabilities, such that the transgene will be deliveredspecifically to a predetermined tissue(s). The AAV capsid is animportant element in determining these tissue-specific targetingcapabilities. Thus an rAAV having a capsid appropriate for the tissuebeing targeted can be selected.

Methods for obtaining rAAVs having a desired capsid protein are wellknown in the art. (See, for example, US 2003/0138772), the contents ofwhich are incorporate herein by reference in their entirety). Typicallythe methods involve culturing a host cell which contains a nucleic acidsequence encoding an AAV capsid protein (e.g., AAV2, AAV5, AAV9) orfragment thereof, as defined herein; a functional rep gene; arecombinant AAV vector composed of, at a minimum, AAV inverted terminalrepeats (ITRs) and a transgene; and sufficient helper functions topermit packaging of the recombinant AAV vector into the AAV capsidproteins.

The components to be cultured in the host cell to package a rAAV vectorin an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,recombinant AAV vector, rep sequences, cap sequences, and/or helperfunctions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein, in the discussion ofregulatory elements suitable for use with the transgene. In stillanother alternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contain the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

The recombinant AAV vector, rep sequences, cap sequences, and helperfunctions required for producing the rAAV of the invention may bedelivered to the packaging host cell in the form of any genetic elementwhich transfers the sequences publicly carried thereon. The selectedgenetic element may be delivered by any suitable method, including thosedescribed herein. The methods used to construct any embodiment of thisinvention are known to those with skill in nucleic acid manipulation andinclude genetic engineering, recombinant engineering, and synthetictechniques. See, e.g., Sambrook et al, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly,methods of generating rAAV virions are well known and the selection of asuitable method is not a limitation on the present invention. See, e.g.,K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No.5,478,745.

The foregoing methods for packaging recombinant vectors in desired AAVcapsids to produce the rAAVs of the invention are not meant to belimiting and other suitable methods will be apparent to the skilledartisan.

The recombinant vector is composed of, at a minimum, a transgene and itsregulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs). It is this recombinant AAV vector which is packaged into acapsid protein and delivered to a selected host cell.

The AAV sequences employed are preferably the cis-acting 5′ and 3′inverted terminal repeat sequences [See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)]. The ITR sequences are about 145 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. [See, e.g., texts such as Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)]. Anexample of such a molecule employed in the present invention is a“cis-acting” plasmid containing the transgene, in which the selectedtransgene sequence and associated regulatory elements are flanked by the5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained fromany known AAV, including presently identified mammalian AAV types. Forexample, the ITRs may be provided by AAV type 1, AAV type 2, AAV type 3,AAV type 4, AAV type 5, AAV 6, other AAV serotypes or parvovirus, e.g.,densovirus.

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes conventional control elementsnecessary which are operably linked to the transgene in a manner whichpermits its transcription, translation and/or expression in a celltransfected with the plasmid vector or infected with the virus producedby the invention. As used herein, “operably linked” sequences includeboth expression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence, miRNAsequence, shRNA sequence, miRNA sponge sequence etc.) and regulatorysequences are said to be “operably” joined when they are covalentlylinked in such a way as to place the expression or transcription of thenucleic acid sequence under the influence or control of the regulatorysequences. If it is desired that the nucleic acid sequences betranslated into a functional protein, two DNA sequences are said to beoperably joined if induction of a promoter in the 5′ regulatorysequences results in the transcription of the coding sequence and if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region to direct the transcription of the codingsequences, or (3) interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a promoter regionwould be operably joined to a nucleic acid sequence if the promoterregion were capable of effecting transcription of that DNA sequence suchthat the resulting transcript might be translated into the desiredprotein or polypeptide. Similarly two or more coding regions areoperably joined when they are linked in such a way that theirtranscription from a common promoter results in the expression of two ormore proteins having been translated in frame. In some embodiments,operably joined coding sequences yield a fusion protein.

The polyadenylation sequence generally is inserted following thetransgene sequences and before the 3′ AAV ITR sequence. A rAAV constructuseful in the present invention may also contain an intron, desirablylocated between the promoter/enhancer sequence and the transgene. Onepossible intron sequence is also derived from SV-40, and is referred toas the SV-40 T intron sequence. Another vector element that may be usedis an internal ribosome entry site (IRES). An IRES sequence is used toproduce more than one polypeptide from a single gene transcript. An IRESsequence would be used to produce a protein that contain more than onepolypeptide chains. Selection of these and other common vector elementsare conventional and many such sequences are available [see, e.g.,Sambrook et al, and references cited therein at, for example, pages 3.183.26 and 16.17 16.27 and Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1989].

The target sites in the mRNA may be in the 5′ UTR, the 3′ UTR or in thecoding region. Interestingly, multiple miRNAs may regulate the same mRNAtarget by recognizing the same or multiple sites. The nucleic acid mayalso comprise a sequence of a target miRNA binding site, or a variantthereof. The presence of multiple miRNA binding sites in mostgenetically identified targets may indicate that the cooperative actionof multiple RISCs provides the most efficient translational inhibition.The target site sequence may comprise a total of 5-100 or 10-60nucleotides. The target site sequence may comprise at least 5nucleotides of the sequence of a target gene binding site.

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of theinvention may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system [WO 98/10088]; theecdysone insect promoter [No et al, Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)], the tetracycline-repressible system [Gossen et al,Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], thetetracycline-inducible system [Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)], the RU486-inducible system [Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)] and therapamycin-inducible system [Magari et al, J. Clin. Invest.,100:2865-2872 (1997)]. Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

Genes encoding small interfering nucleic acids, e.g., miRNAs, miRNAInhibitors, may be expressed from any appropriate promoter, including,but not limited to, a RNA polymerase II promoter, a RNA polymerase IIIpromoter, or a combination thereof. A small interfering nucleic acid,e.g., miRNA, miRNA Inhibitor, may be operably joined with a combinationof promoters including, for example, a combination of a RNA polymeraseII promoter and a RNA III promoter.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

In preferred embodiments, the regulatory sequences imparttissue-specific gene expression capabilities. In some cases, thetissue-specific regulatory sequences bind tissue-specific transcriptionfactors that induce transcription in a tissue specific manner. Suchtissue-specific regulatory sequences (e.g., promoters, enhancers, etc.)are well known in the art. Exemplary tissue-specific regulatorysequences include, but are not limited to the following tissue specificpromoters: a liver-specific thyroxin binding globulin (TBG) promoter, ainsulin promoter, a glucagon promoter, a somatostatin promoter, apancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, acreatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, aα-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT)promoter. Other exemplary promoters include Beta-actin promoter,hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9(1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. GeneTher., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol.Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al.,J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cellreceptor α-chain promoter, neuronal such as neuron-specific enolase(NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15(1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc.Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgfgene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among otherswhich will be apparent to the skilled artisan.

In some embodiments, the transgene is a nucleic acid sequence,heterologous to the vector sequences, which encodes a polypeptide,protein, RNA molecule (e.g., miRNA, miRNA sponge) or other gene product,of interest. The nucleic acid coding sequence is operatively linked toregulatory components in a manner which permits transgene transcription,translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the resulting vector will be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, and others wellknown in the art. When associated with regulatory elements which drivetheir expression, the reporter sequences, provide signals detectable byconventional means, including enzymatic, radiographic, colorimetric,fluorescence or other spectrographic assays, fluorescent activating cellsorting assays and immunological assays, including enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) andimmunohistochemistry. For example, where the marker sequence is the LacZgene, the presence of the vector carrying the signal is detected byassays for β-galactosidase activity. Where the transgene is greenfluorescent protein or luciferase, the vector carrying the signal may bemeasured visually by color or light production in a luminometer. Suchreporters can, for example, be useful in verifying the tissue-specifictargeting capabilities and tissue specific promoter regulatory activityof an rAAV.

The invention in some aspects, provide methods for producing somatictransgenic animal models through the targeted destruction of specificcell types. For example, models of type 1 diabetes can be produced bythe targeted destruction of pancreatic Beta-islets. In other examples,the targeted destruction of specific cell types can be used to evaluatethe role of specific cell types on human disease. In this regard,transgenes that encode cellular toxins (e.g., diphtheria toxin A (DTA))or pro-apoptotic genes (NTR, Box, etc.) can be useful as transgenes forfunctional ablation of specific cell types. Other exemplary transgenes,whose products kill cells are embraced by the methods disclosed hereinand will be apparent to one of ordinary skill in the art.

The long term over expression or knockdown (e.g., by miRNA and/or miRNAsponge) of genes in specific target tissues can disturb normal metabolicbalance and establish a pathological state, thereby producing an animalmodel of diseases such as cancer. The invention in some aspects,provides methods for producing somatic transgenic animal models to studythe long-term effects of over-expression or knockdown of potentialoncogenes and other genes to study tumorigenesis and gene function inthe targeted tissues. Useful transgene products include proteins thatare known to be associated with cancer and small interfering nucleicacids inhibiting the expression of such proteins. The following is anon-limiting list of gene known to be associated with the development ofcancer (e.g., oncogenes and tumor suppressors) and nucleic acidsencoding the products of these genes and their homologues and encodingsmall interfering nucleic acids (e.g., shRNAs, miRNAs, miRNA sponges)that inhibit the expression of these genes and their homologues areuseful as transgenes in certain embodiments of the methods: AARS, ABCB1,ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2, AKT1, ALB,ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4,ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2, BHLHB2, BLMH, BRAF, BRCA1,BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1, CBFB, CBLB, CCL2, CCND1,CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25,CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9, CDKL1, CDKN1A, CDKN1B,CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A, CIB1,CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1, COL6A3, COX6C, COX7A2,CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1, CTPS, CTSC, CTSD,CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8, DLG3, DVL1, DVL3,E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2, ERBB3, ERBB4, ERCC3,ETV1, ETV3, ETV6, F2R, FASTK, FBN1, FBN2, FES, FGFR1, FGR, FKBP8, FN1,FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB, FTL, FZD2, FZD5, FZD9,G22P1, GAS6, GCN5L2, GDF15, GNA13, GNAS, GNB2, GNB2L1, GPR39, GRB2,GSK3A, GSPT1, GTF2I, HDAC1, HDGF, HMMR, HPRT1, HRB, HSPA4, HSPA5, HSPA8,HSPB1, HSPH1, HYAL1, HYOU1, ICAM1, ID1, ID2, IDUA, IER3, IFITM1, IGF1R,IGF2R, IGFBP3, IGFBP4, IGFBP5, IL1B, ILK, ING1, IRF3, ITGA3, ITGA6,ITGB4, JAK1, JARID1A, JUN, JUNB, JUND, K-ALPHA-1, KIT, KITLG, KLK10,KPNA2, KRAS2, KRT18, KRT2A, KRT9, LAMB1, LAMP2, LCK, LCN2, LEP, LITAF,LRPAP1, LTF, LYN, LZTR1, MADH1, MAP2K2, MAP3K8, MAPK12, MAPK13,MAPKAPK3, MAPRE1, MARS, MAS1, MCC, MCM2, MCM4, MDM2, MDM4, MET, MGST1,MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA, MSH2, MSH6, MT3, MYB,MYBL1, MYBL2, MYC, MYCL1, MYCN, MYD88, MYL9, MYLK, NEO1, NF1, NF2,NFKB1, NFKB2, NFSF7, NID, NINJ1, NMBR, NME1, NME2, NME3, NOTCH1, NOTCH2,NOTCH4, NPM1, NQO1, NR1D1, NR2F1, NR2F6, NRAS, NRG1, NSEP1, OSM, PA2G4,PABPC1, PCNA, PCTK1, PCTK2, PCTK3, PDGFA, PDGFB, PDGFRA, PDPK1, PEA15,PFDN4, PFDN5, PGAM1, PHB, PIK3CA, PIK3CB, PIK3CG, PIM1, PKM2, PKMYT1,PLK2, PPARD, PPARG, PPIH, PPP1CA, PPP2R5A, PRDX2, PRDX4, PRKAR1A,PRKCBP1, PRNP, PRSS15, PSMA1, PTCH, PTEN, PTGS1, PTMA, PTN, PTPRN,RAB5A, RAC1, RAD50, RAF1, RALBP1, RAP1A, RARA, RARB, RASGRF1, RB1,RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA, RHOB, RHOC,RHOD, RIPK1, RPN2, RPS6KB1, RRM1, SARS, SELENBP1, SEMA3C, SEMA4D, SEPP1,SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVA TP53, SKI, SKIL,SLC16A1, SLC1A4, SLC20A1, SMO, SMPD1, SNAI2, SND1, SNRPB2, SOCS1, SOCS3,SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1, STAT2, STAT3, STAT5B,STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP1, TFDP2, TGFA,TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, TK1, TLE1,TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TNK1,TOB1, TP53, TP53BP2, TP53I3, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP,TSG101, TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL,VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1, XRCC1, YES1, YWHAB,YWHAZ, ZAP70, and ZNF9.

Other useful transgene products include proteins that are known to beassociated with apoptosis. The following is a non-limiting list of geneknown to be associated with apoptosis and nucleic acids encoding theproducts of these genes and their homologues and encoding smallinterfering nucleic acids (e.g., shRNAs, miRNAs, miRNA sponges) thatinhibit the expression of these genes and their homologues are useful astransgenes in certain embodiments of the methods: RPS27A, ABL1, AKT1,APAF1, BAD, BAG1, BAG3, BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1, BCL2L1,BCL2L10, BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK,NAIP, BIRC2, BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2,BNIP3, BNIP3L, BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2, NOD1,CARD6, CARDS, CARDS, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5,CASP6, CASP7, CASP8, CASP9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2,DFFA, DFFB, FADD, GADD45A, GDNF, HRK, IGF1R, LTA, LTBR, MCL1, NOL3,PYCARD, RIPK1, RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D,TNFRSF11B, TNFRSF12A, TNFRSF14, TNFRSF19, TNFRSF1A, TNFRSF1B, TNFRSF21,TNFRSF25, CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, TNFSF18,CD40LG, FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD,TRAF1, TRAF2, TRAF3, TRAF4, and TRAF5.

In some embodiments, the sequence of the coding region of a transgene ismodified. In some cases, the modification alters the function of theproduct encoded by the transgene. The effect of the modification canthen be studied in vivo by generating a somatic transgenic animal modelusing the methods disclosed herein. In some embodiments, modification ofthe sequence of coding region is a nonsense mutation that results in afragment (e.g., a truncated version). In other cases, the modificationis a missense mutation that results in an amino acid substitution. Othermodifications are possible and will be apparent to the skilled artisan.

The skilled artisan will also realize that in the case of transgenesencoding proteins or polypeptides, that conservative amino acidsubstitutions may be made in transgenes to provide functionallyequivalent variants, or homologs of the proteins or polypeptides. Insome aspects the invention embraces sequence alterations that result inconservative amino acid substitution of a transgene. As used herein, aconservative amino acid substitution refers to an amino acidsubstitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references that compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D. Therefore, one can make conservativeamino acid substitutions to the amino acid sequence of the proteins andpolypeptides disclosed herein.

miRNAs and other small interfering nucleic acids regulate geneexpression via target RNA transcript cleavage/degradation ortranslational repression of the target messenger RNA (mRNA). miRNAs arenatively expressed, typically as final 19-25 non-translated RNAproducts. miRNAs exhibit their activity through sequence-specificinteractions with the 3′ untranslated regions (UTR) of target mRNAs.These endogenously expressed miRNAs form hairpin precursors which aresubsequently processed into an miRNA duplex, and further into a “mature”single stranded miRNA molecule. This mature miRNA guides a multiproteincomplex, miRISC, which identifies target 3′ UTR regions of target mRNAsbased upon their complementarity to the mature miRNA. The invention insome aspects relates to the study of microRNA function in specifictarget tissues and cell types. Thus, useful transgene products includemiRNAs. The following is a non-limiting list of miRNA genes; theproducts of these genes and their homologues are useful as transgenes oras targets for small interfering nucleic acids (e.g., miRNA sponges,antisense oligonucleotides) in certain embodiments of the methods:hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c,hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*,hsa-let-7f, hsa-let-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*,hsa-let-7i, hsa-let-7i*, hsa-miR-1, hsa-miR-100, hsa-miR-100*,hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*,hsa-miR-106a, hsa-miR-106a*, hsa-miR-106b, hsa-miR-106b*, hsa-miR-107,hsa-miR-10a, hsa-miR-10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178,hsa-miR-1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183,hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR-1201,hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206,hsa-miR-1207-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122,hsa-miR-122*, hsa-miR-1224-3p, hsa-miR-1224-5p, hsa-miR-1225-3p,hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-1226*, hsa-miR-1227,hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233,hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124,hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246,hsa-miR-1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251,hsa-miR-1252, hsa-miR-1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b,hsa-miR-1256, hsa-miR-1257, hsa-miR-1258, hsa-miR-1259, hsa-miR-125a-3p,hsa-miR-125a-5p, hsa-miR-125b, hsa-miR-125b-1*, hsa-miR-125b-2*,hsa-miR-126, hsa-miR-126*, hsa-miR-1260, hsa-miR-1261, hsa-miR-1262,hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267,hsa-miR-1268, hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272,hsa-miR-1273, hsa-miR-12′7-3p, hsa-miR-1274a, hsa-miR-1274b,hsa-miR-1275, hsa-miR-127-5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278,hsa-miR-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282,hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287,hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291,hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295,hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299,hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304,hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a,hsa-miR-130a*, hsa-miR-130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*,hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa-miR-1324, hsa-miR-133a,hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b,hsa-miR-135b*, hsa-miR-136, hsa-miR-136*, hsa-miR-137, hsa-miR-138,hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p,hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141, hsa-miR-141*,hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144,hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa-miR-146a*,hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-147, hsa-miR-147b,hsa-miR-148a, hsa-miR-148a*, hsa-miR-148b, hsa-miR-148b*, hsa-miR-149,hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-3p, hsa-miR-151-5p,hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154*, hsa-miR-155,hsa-miR-155*, hsa-miR-15a, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*,hsa-miR-16, hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17, hsa-miR-17*,hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b,hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-182*,hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*,hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*,hsa-miR-187, hsa-miR-187*, hsa-miR-188-3p, hsa-miR-188-5p, hsa-miR-18a,hsa-miR-18a*, hsa-miR-18b, hsa-miR-18b*, hsa-miR-190, hsa-miR-190b,hsa-miR-191, hsa-miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p,hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b*, hsa-miR-194, hsa-miR-194*,hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b,hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p,hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19a*, hsa-miR-19b, hsa-miR-19b-1*,hsa-miR-19b-2*, hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b,hsa-miR-200b*, hsa-miR-200c, hsa-miR-200c*, hsa-miR-202, hsa-miR-202*,hsa-miR-203, hsa-miR-204, hsa-miR-205, hsa-miR-206, hsa-miR-208a,hsa-miR-208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20b, hsa-miR-20b*,hsa-miR-21, hsa-miR-21*, hsa-miR-210, hsa-miR-211, hsa-miR-212,hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b,hsa-miR-217, hsa-miR-218, hsa-miR-218-1*, hsa-miR-218-2*,hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22,hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221,hsa-miR-221*, hsa-miR-222, hsa-miR-222*, hsa-miR-223, hsa-miR-223*,hsa-miR-224, hsa-miR-23a, hsa-miR-23a*, hsa-miR-23b, hsa-miR-23b*,hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*,hsa-miR-26a, hsa-miR-26a-1*, hsa-miR-26a-2*, hsa-miR-26b, hsa-miR-26b*,hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p,hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298,hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b,hsa-miR-29b-1*, hsa-miR-29b-2*, hsa-miR-29c, hsa-miR-29c*, hsa-miR-300,hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b,hsa-miR-302b*, hsa-miR-302c, hsa-miR-302c*, hsa-miR-302d, hsa-miR-302d*,hsa-miR-302e, hsa-miR-302f, hsa-miR-30a, hsa-miR-30a*, hsa-miR-30b,hsa-miR-30b*, hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30d,hsa-miR-30d*, hsa-miR-30e, hsa-miR-30e*, hsa-miR-31, hsa-miR-31*,hsa-miR-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c,hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p,hsa-miR-324-5p, hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-329,hsa-miR-330-3p, hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331-5p,hsa-miR-335, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p,hsa-miR-338-3p, hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5p,hsa-miR-33a, hsa-miR-33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340,hsa-miR-340*, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346,hsa-miR-34a, hsa-miR-34a*, hsa-miR-34b, hsa-miR-34b*, hsa-miR-34c-3p,hsa-miR-34c-5p, hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3p,hsa-miR-362-5p, hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367,hsa-miR-367*, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370,hsa-miR-371-3p, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*,hsa-miR-374a, hsa-miR-374a*, hsa-miR-374b, hsa-miR-374b*, hsa-miR-375,hsa-miR-376a, hsa-miR-376a*, hsa-miR-376b, hsa-miR-376c, hsa-miR-377,hsa-miR-377*, hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*,hsa-miR-380, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383,hsa-miR-384, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411,hsa-miR-411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p,hsa-miR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*,hsa-miR-429, hsa-miR-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*,hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a,hsa-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452,hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p,hsa-miR-455-5p, hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484,hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR-486-3p, hsa-miR-486-5p,hsa-miR-487a, hsa-miR-487b, hsa-miR-488, hsa-miR-488*, hsa-miR-489,hsa-miR-490-3p, hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p,hsa-miR-492, hsa-miR-493, hsa-miR-493*, hsa-miR-494, hsa-miR-495,hsa-miR-496, hsa-miR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p,hsa-miR-499-5p, hsa-miR-500, hsa-miR-500*, hsa-miR-501-3p,hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503,hsa-miR-504, hsa-miR-505, hsa-miR-505*, hsa-miR-506, hsa-miR-507,hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509-3-5p, hsa-miR-509-3p,hsa-miR-509-5p, hsa-miR-510, hsa-miR-511, hsa-miR-512-3p,hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513a-5p, hsa-miR-513b,hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p, hsa-miR-515-5p,hsa-miR-516a-3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517*,hsa-miR-517a, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a-3p,hsa-miR-518a-5p, hsa-miR-518b, hsa-miR-518c, hsa-miR-518c*,hsa-miR-518d-3p, hsa-miR-518d-5p, hsa-miR-518e, hsa-miR-518e*,hsa-miR-518f, hsa-miR-518f*, hsa-miR-519a, hsa-miR-519b-3p,hsa-miR-519c-3p, hsa-miR-519d, hsa-miR-519e, hsa-miR-519e*,hsa-miR-520a-3p, hsa-miR-520a-5p, hsa-miR-520b, hsa-miR-520c-3p,hsa-miR-520d-3p, hsa-miR-520d-5p, hsa-miR-520e, hsa-miR-520f,hsa-miR-520g, hsa-miR-520h, hsa-miR-521, hsa-miR-522, hsa-miR-523,hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p,hsa-miR-526b, hsa-miR-526b*, hsa-miR-532-3p, hsa-miR-532-5p,hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR-542-3p, hsa-miR-542-5p,hsa-miR-543, hsa-miR-544, hsa-miR-545, hsa-miR-545*, hsa-miR-548a-3p,hsa-miR-548a-5p, hsa-miR-548b-3p, hsa-miR-548b-5p, hsa-miR-548c-3p,hsa-miR-548c-5p, hsa-miR-548d-3p, hsa-miR-548d-5p, hsa-miR-548e,hsa-miR-548f, hsa-miR-548g, hsa-miR-548h, hsa-miR-548i, hsa-miR-548j,hsa-miR-548k, hsa-miR-548l, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o,hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hsa-miR-551a,hsa-miR-551b, hsa-miR-551b*, hsa-miR-552, hsa-miR-553, hsa-miR-554,hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558,hsa-miR-559, hsa-miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564,hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570,hsa-miR-571, hsa-miR-572, hsa-miR-573, hsa-miR-574-3p, hsa-miR-574-5p,hsa-miR-575, hsa-miR-576-3p, hsa-miR-576-5p, hsa-miR-577, hsa-miR-578,hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582-3p, hsa-miR-582-5p,hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587,hsa-miR-588, hsa-miR-589, hsa-miR-589*, hsa-miR-590-3p, hsa-miR-590-5p,hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-593*, hsa-miR-595,hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600,hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605,hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610,hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615-3p,hsa-miR-615-5p, hsa-miR-616, hsa-miR-616*, hsa-miR-617, hsa-miR-618,hsa-miR-619, hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623,hsa-miR-624, hsa-miR-624*, hsa-miR-625, hsa-miR-625*, hsa-miR-626,hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629*,hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR-633, hsa-miR-634,hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639,hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644,hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649,hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR-653, hsa-miR-654-3p,hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658,hsa-miR-659, hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663,hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668,hsa-miR-671-3p, hsa-miR-671-5p, hsa-miR-675, hsa-miR-7, hsa-miR-708,hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2*, hsa-miR-720, hsa-miR-744,hsa-miR-744*, hsa-miR-758, hsa-miR-760, hsa-miR-765, hsa-miR-766,hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-768-3p, hsa-miR-768-5p,hsa-miR-769-3p, hsa-miR-769-5p, hsa-miR-770-5p, hsa-miR-802,hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p,hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-miR-877*,hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p, hsa-miR-886-5p,hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-889, hsa-miR-890,hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9,hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR-922, hsa-miR-923,hsa-miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b,hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR-933, hsa-miR-934,hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, hsa-miR-939,hsa-miR-940, hsa-miR-941, hsa-miR-942, hsa-miR-943, hsa-miR-944,hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR-98, hsa-miR-99a,hsa-miR-99a*, hsa-miR-99b, and hsa-miR-99b*. In some embodiments, one ormore bindings sites for one or more of the foregoing miRNAs areincorporated in a transgene, e.g., a transgene delivered by a rAAVvector, to inhibit the expression of the transgene in one or moretissues of an animal harboring the transgene. The skilled artisan willappreciate that binding sites may be selected to control the expressionof a trangene in a tissue specific manner. For example, binding sitesfor the liver-specific miR-122 may be incorporated into a transgene toinhibit expression of that transgene in the liver.

An miRNA inhibits the function of the mRNAs it targets and, as a result,inhibits expression of the polypeptides encoded by the mRNAs. Thus,blocking (partially or totally) the activity of the miRNA (e.g.,silencing the miRNA) can effectively induce, or restore, expression of apolypeptide whose expression is inhibited (derepress the polypeptide).In one embodiment, derepression of polypeptides encoded by mRNA targetsof an miRNA is accomplished by inhibiting the miRNA activity in cellsthrough any one of a variety of methods. For example, blocking theactivity of an miRNA can be accomplished by hybridization with a smallinterfering nucleic acid (e.g., antisense oligonucleotide, miRNA sponge)that is complementary, or substantially complementary to, the miRNA,thereby blocking interaction of the miRNA with its target mRNA. As usedherein, an small interfering nucleic acid that is substantiallycomplementary to an miRNA is one that is capable of hybridizing with anmiRNA, and blocking the miRNA's activity. In some embodiments, an smallinterfering nucleic acid that is substantially complementary to an miRNAis an small interfering nucleic acid that is complementary with themiRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, or 18 bases. In some embodiments, an small interfering nucleic acidsequence that is substantially complementary to an miRNA, is an smallinterfering nucleic acid sequence that is complementary with the miRNAat, at least, one base. MicroRNA inhibitors, e.g., miRNA sponges, can beexpressed in cells from transgenes (Ebert, M. S. Nature Methods, EpubAug. 12, 2007). These miRNA sponges specifically inhibit miRNAs througha complementary heptameric seed sequence and an entire family of miRNAscan be silenced using a single sponge sequence. Other transgenic methodsfor silencing miRNA function (derepression of miRNA targets) in cellswill be apparent to one of ordinary skill in the art.

Other suitable transgenes may be readily selected by one of skill in theart provided that they are useful for creating animal models oftissue-specific pathological state and/or disease.

A “miR Inhibitor” or “miRNA Inhibitor” is an agent that blocks miRNAexpression and/or processing. For instance, these molecules include butare not limited to microRNA antagonists, microRNA specific antisense,microRNA sponges, and microRNA oligonucleotides (double-stranded,hairpin, short oligonucleotides) that inhibit miRNA interaction with aDrosha complex. MicroRNA inhibitors, e.g., miRNA sponges, can beexpressed in cells from transgenes (Ebert, M. S. Nature Methods, EpubAug. 12, 2007). The invention encompasses the use of microRNA sponges,or other miR Inhibitors, with the AAVs. These microRNA spongesspecifically inhibit miRNAs through a complementary heptameric seedsequence. An entire family of miRNAs can be silenced using a singlesponge sequence. Other methods for silencing miRNA function(derepression of miRNA targets) in cells will be apparent to one ofordinary skill in the art.

The rAAVs may be delivered to host animal in compositions according toany appropriate methods known in the art. The rAAV, preferably suspendedin a physiologically compatible carrier (i.e., in a composition), may beadministered to a host animal, such as a mouse, rat, cat, dog, sheep,rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, ora non-human primate (e.g, Macaque). In some embodiments a host animaldoes not include a human.

The compositions of the invention may comprise an rAAV alone, or incombination with one or more other viruses (e.g., a second rAAV encodinghaving one or more different transgenes). In some embodiments, acompositions comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more differentrAAV each having one or more different transgenes.

Suitable carriers may be readily selected by one of skill in the art inview of the indication for which the transfer virus is directed. Forexample, one suitable carrier includes saline, which may be formulatedwith a variety of buffering solutions (e.g., phosphate buffered saline).Other exemplary carriers include sterile saline, lactose, sucrose,calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesameoil, and water. The selection of the carrier is not a limitation of thepresent invention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The rAAVS are administered in sufficient amounts to transfect the cellsof a desired tissue and to provide sufficient levels of gene transferand expression without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected organ (e.g., intraportaldelivery to the liver), oral, inhalation (including intranasal andintratracheal delivery), intraocular, intravenous, intramuscular,subcutaneous, intradermal, intratumoral, and other parental routes ofadministration. Routes of administration may be combined, if desired.

An effective amount of an rAAV is an amount sufficient to target infectan animal, target a desired tissue, and produce a stable somatictransgenic animal model. The effective amount will depend primarily onfactors such as the species, age, weight, health of the animal, and thetissue to be targeted, and may thus vary among animal and tissue. Forexample, a effective amount of the rAAV viral vector is generally in therange of from about 1 ml to about 100 ml of solution containing fromabout 10⁹ to 10¹⁶ genome copies. In some cases, a dosage between about10¹¹ to 10¹² rAAV genome copies is appropriate. In certain preferredembodiments, 10¹² rAAV genome copies is effective to target heart,liver, and pancreas tissues. In some cases, stable transgenic animalsare produced by multiple doses of an rAAV.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound in eachtherapeutically-useful composition may be prepared is such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the AAVvector-based therapeutic constructs in suitably formulatedpharmaceutical compositions disclosed herein either subcutaneously,intraopancreatically, parenterally, intravenously, intramuscularly,intrathecally, or even orally, intraperitoneally, or by nasalinhalation, including those modalities as described in U.S. Pat. Nos.5,543,158; 5,641,515 and 5,399,363 (each specifically incorporatedherein by reference in its entirety). One preferred mode ofadministration is by portal vein. Solutions of the active compounds asfreebase or pharmacologically acceptable salts may be prepared insterile water and may also suitably mixed with one or more surfactants,such as hydroxypropylcellulose. Dispersions may also be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations containa preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the activeAAV vector in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The AAV vector compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present invention intosuitable host cells. In particular, the rAAV vector delivered trangenesmay be formulated for delivery either encapsulated in a lipid particle,a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trails examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the AAV vector-basedpolynucleotide may be used. Nanocapsules can generally entrap compoundsin a stable and reproducible way. To avoid side effects due tointracellular polymeric overloading, such ultrafine particles (sizedaround 0.1 μm) should be designed using polymers able to be degraded invivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meetthese requirements are contemplated for use.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe rAAV vector compositions to a host. Sonophoresis (ie., ultrasound)has been used and described in U.S. Pat. No. 5,656,016 as a device forenhancing the rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

The agents described herein may, in some embodiments, be assembled intopharmaceutical or diagnostic or research kits to facilitate their use intherapeutic, diagnostic or research applications. A kit may include oneor more containers housing the components of the invention andinstructions for use. Specifically, such kits may include one or moreagents described herein, along with instructions describing the intendedapplication and the proper use of these agents. In certain embodimentsagents in a kit may be in a pharmaceutical formulation and dosagesuitable for a particular application and for a method of administrationof the agents. Kits for research purposes may contain the components inappropriate concentrations or quantities for running variousexperiments.

For instance a kit is provided for identifying an AAV serotype and/orfor distinguishing novel AAV from known AAV. Other kits provided hereinare for detecting the presence of a known or unknown AAV in a sample.Yet another kit of the invention involves the use of a sponge sequencefor analyzing miRNA function.

The kit may be designed to facilitate use of the methods describedherein by researchers and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for animal administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may bein the form of a liquid, gel or solid (powder). The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to ananimal, such as a syringe, topical application devices, or iv needletubing and bag, particularly in the case of the kits for producingspecific somatic animal models.

Kits are useful in some instances for rescuing contaminates by helperAAV infections and/or detection of latent virus that istranscriptionally active. Examples of such are shown in the examples.

The containers of the kit may house, for instance, any one or more ofthe following: at least one RNA detection component, at least one primerthat has substantial homology with a nucleic acid sequence that is about90% conserved between at least two AAV serotypes, at least one primerthat is substantially complementary to a nucleic acid sequencecorresponding to a 5′ or 3′ untranslated region of an AAV transcriptsuch as a transcript encoding a rep and/or cap gene, a set of PCRprimers specific for a signature region of the AAV nucleic acidsequence, a set of PCR primers specific for the full-length AAV capsidtranscript (i.e., the p40 initiated transcript), two or more additionalsets of primers, as described herein, and/or PCR probes, a primer havinga sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 4.

The kits may also include reagents for Reverse transcription componentsthat may include the following components: (a) at least one primer; (b)a Reverse Transcriptase (e.g., a Superscript); (c) nucleotides (e.g.,dNTPs); and (d) RT buffer. In some embodiments, the at least one primeris complementary to a portion of an AAV cDNA sequence. In someembodiments, the at least one primer is an OligodT primer. In someembodiments, the kits further comprise reagents for PCR components thatmay include the following components: (a) at least one primer; (b) athermostable polymerase (e.g., a Taq polymerase); (c) nucleotides (e.g.,dNTPs); and (d) PCR buffer. In some embodiments, the at least one primeris complementary to a portion of an AAV cDNA sequence. In someembodiments, the kits comprise a DNA isolation kit (e.g., Oragene,OG-100) and/or an RNA isolation kit (e.g., oligodT-cellulose columns).

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kit may besterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kit may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration etc.

The instructions included within the kit may involve methods fordetecting a latent AAV in a cell. In addition, kits of the invention mayinclude, instructions, a negative and/or positive control, containers,diluents and buffers for the sample, sample preparation tubes and aprinted or electronic table of reference AAV sequence for sequencecomparisons.

Examples Example 1: Micro RNA Regulated Tissue Specific Transduction byrAAV Vector

Micro RNAs (miRNAs) are small RNA species with approximately 18-24nucleotides in size. They regulate gene expression bypost-transcriptional silencing through pairing to the partiallycomplementary sites located in the 3′ UTRs of transcripts of the targetgenes. Regulation of transgene expression in a cell type specific mannerby endogenous miRNA was first introduced into lentiviral vectors tosuppress transgene expression in hematopoietic cells and abolishtransgene immunity. Many of the novel AAV vectors that were discoveredrecently demonstrate strong hepatic tissue tropism while exhibitingunique transduction profiles in other tissues. Depending on theapplications, intended target tissue and gene of interest to bedelivered, inadvertent transduction in liver and other tissues may leadto untoward outcomes which should be avoided. Previously, we have shownthat systemic delivery of AAV9 vector can efficiently transduce liver,heart, pancreas, skeletal and diaphragm muscles with an expressioncassette driven by an ubiquitous promoter such as chicken β-actinprompter (CB). We also demonstrated that tissue specific expressionaltargeting in myocardium and islet could be accomplished by introducingtissue/cell type specific promoter into the expression cassette,although levels of transgene expression in the target tissues weresomewhat compromised and some off-target expression in liver wasdetected in the high dose regimen (10¹² GC/mouse).

In the present study, we explored use of endogenous miRNAs to regulatetissue specific transduction profiles of rAAV in mouse models. In aproof-of-concept experiment, we tested the potency of miRNA mediatedtransgene silencing in mouse liver by introducing the binding sites ofmi122, the most abundant micro RNA species in the liver, to the TBGpromoter (the strongest liver specific promoter in our lab) driven nLacZexpression cassette. The rAAV vectors with and without mi122 bindingsites were packaged with AAV9 capsid and delivered at high dose (1×10¹²GC/mouse) to mouse liver via IV injections. X-gal histochemical stainingof the liver sections at four weeks after gene transfer revealed almostcomplete suppression of nLacZ transduction in the liver of animalsinjected with the construct containing mi122 binding sites, suggestingendogenous mi122 RNA mediated transgene silencing in mouse liver ishighly effective. The same vector design was then applied to the CbnLacZconstruct for a similar comparison of expressional targeting for mouseliver, heart and some other tissues. The mi122 binding site bearingconstruct led to a 100% suppression of liver transduction without anyimpact on the heart and pancreas transduction. Other manipulations inendogenous microRNA mediated regulation of transgene expression havealso been investigated. These include the dose response of genesilencing to the numbers of miRNA binding sites, simultaneous transgenesuppression in multiple tissues and cell types through different miRNAspecies, and rescue of transgene expression from miRNA mediated genesilencing by miRNA sponge, etc.

The ability of rAAVs to deliver transgenes that are susceptible to miRNAtargeting and degradation was assessed. AAV−miRBS was constructed (FIGS.11 and 12). AAV-miRBS is a transgene having a lacZ coding sequence, amiR-122 binding site and was determined to be susceptible to miR-122based cleavage. (FIG. 1A). Expression of AAV−miRBS, which containedbinding sites for miR-122 did not significantly affect miR-122expression or Cyclin G1 expression (mRNA or protein), which is a targetof miR-122. (FIG. 1B).

A recombinant AAV, rAAV9.CB.nLacZ.3xmiR122, was constructed thatcontains a LacZ coding sequence flanked by 5′ and 3′ inverted terminalrepeat sequences. rAAV.CB.nLacZ.3xmi122 also has a ubiquitous EM7promoter, CMV immediate early enhancer and CB promoter sequences, andthree miR-122 binding sites in the 3′ UTR. (FIG. 2A.) Mice were infectedwith the rAAVs at high dose (1×10¹² GC/mouse) and LacZ expression wasevaluated in the heart and liver tissue. Expression ofrAAV.CB.nLacZ.3xmiR122 is detectable by B-Galactosidase staining inheart tissue but not liver tissue, where miR-122 is expressed. (FIG.2B.) Control rAAV that does not contain miR122 binding sites isexpressed in both heart and liver tissue. (FIG. 2B.)

Recombinant AAVs were produced that express a LacZ reporter genecontrolled by a chicken beta actin promoter, each having miR-122 bindingsites and/or miR-1 binding sites in its 3′ UTR. Control rAAVs were alsoproduced that express a LacZ reporter gene controlled by a chicken betaactin promoter without miRNA binding sites. Mice were infected with therAAVs at high dose (1×10¹² GC/mouse) and LacZ expression was evaluatedby X-Gal staining of heart, pancreas and liver tissue sections. ControlrAAVs produced high levels of LacZ expression in heart and liver tissue.(FIG. 3, first row.) rAAVs with a single miR-122 site in the 3′-UTRproduced high levels of LacZ reporter gene expression in heart tissuebut not pancreas or liver tissue, indicating that endogenous miR-122inhibited LacZ expression in the liver. (FIG. 3, second row.) rAAVs witha single (or three) miR-1 site(s) in the 3′-UTR produced high levels ofLacZ reporter gene expression in liver tissue but not pancreas or hearttissue, indicating that endogenous miR-1 inhibited LacZ expression inthe heart. (FIG. 3, third and fourth rows.) rAAVs with a single miR-1site and a single miR-122 site in the 3′-UTR did not produce significantlevels of LacZ reporter gene expression in any of the three tissues.(FIG. 3, fifth row.) This result indicates that endogenous miR-1inhibited LacZ expression in the heart, that endogenous miR-122inhibited LacZ expression in the heart, and that multiple differentmiRNA binding sites can be combined to inhibit expression in multipledifferent tissues.

Example 2: rAAV Mediated Delivery of Target Specific Micro RNA Spongesfor Study of Micro-RNA Function in Mouse Models

Micro RNAs (miRNAs) are approximately 18-24 nucleotide long small RNAspecies. They regulate most of cellular processes includingdifferentiation, apoptosis, proliferation and maintain cell and tissueidentify. These functions are accomplished by post-transcriptionalsilencing by pairing to the partially complementary sites located in the3′ UTRs of transcripts of the target genes. Changes in miRNA expressionprofiles could be implicated in human diseases. It has been predictedthat at least a thousand of miRNA species exist in human. However,biological functions of the majority of miRNAs in mammals are poorlyunderstood. One of the approaches to study miRNA functions is to knockdown their expression and examine resulting biological consequences. Thestrategies for miRNA knock-down include miRNA sponge, target masking anderase, which modifies miRNA expression profiles by either scavengingmiRNA or blocking miRNA binding to the target sequences. These haveproven to be effective in primarily in vitro studies. Use of thosedesigns to study miRNA functions in vivo has been hindered by lack ofefficient delivery tools for sustained alternation of miRNA profiles.

Adeno-associated virus (AAV) is a small and non-pathogenic, singlestranded DNA virus. Recent advancement in (AAV) vectorology hasgenerated a battery of novel AAV serotype vectors that are capable ofachieving efficient and stable gene transfer to major target tissues,which makes rAAV an ideal tool for in vivo gene transfer. In an attemptto investigate miRNA functions in vivo, we utilized rAAV platform forefficiently delivering target specific miRNA sponges to mouse models. Ina proof-of-concept study, we selected miR-122 and Let-7 as the targets.MiR-122 is the most abundant miRNA in liver and plays important roles inregulating lipid metabolism. Let-7 is highly expressed in earlydevelopment and tumor cells. It has been implicated in transformationand tumorigenesis. We incorporate bulged binding sites for those twomiRNA species into the 3′ UTR of the fire fly luciferase for packagingwith AAV9 capsid. Transcription of luciferase gene and bulged miRNAbinding sequences is directed by either chicken-β-actin promoter orliver specific promoter TBG. The vector genomes also carries a GFPreporter gene cassette that is not regulated by miRNA to serve as amicroscopic tracer for gene transfer. Vector constructs with wild typeand mutant miRNA sponges are delivered to mice for side by sidecomparison. The animals are monitored for luciferase expression alive todocument translational suppression by wild type miRNA sponges. Lipidprofiles of the animals received mi122 sponges are scrutinized. Serumlevels of α-fetal proteins, tumor incidence and other gross pathologyare documented in the animals injected with Let-7 sponges. In addition,animals are sacrificed at different time points. Total miRNA profilesand expression level of dicer RNA are analyzed in the target tissues.

MicroRNA sponges are competitive inhibitors of microRNAs. MicroRNAsponge transcripts may contain multiple, tandem binding sites to amicroRNA of interest. In some cases, sponges can derepress microRNAtargets at least as strongly as chemically modified antisenseoligonucleotides against microRNAs. Sponges specifically inhibitmicroRNAs with a complementary heptameric seed, such that a singlesponge can be used to block an entire microRNA seed family. Sponges maybe expressed from a RNA polymerase II promoter (Pol II)-driven and maybe combined with a reporter gene (e.g., luciferase, EGFP, etc.) foridentification and sorting of sponge-treated cells. (See, e.g., Ebert MS, Neilson J R, Sharp P A. Nature Methods. 2007, 4(9):721-726.) Theskilled artisan will appreciate that the design of miRNA expressionconstructs involves an assessment of various factors including promoterselection, e.g., a Polymerase III promoter, e.g., a U6 promoter, versusPolymerase II promoter, binding site design, e.g., perfect match versusbulge, and seed sequence selection, e.g., a sponge with an appropriatelydesigned seed sequence may universally knock-down an entire miRNA seedfamily. For example, binding sites for a particular microRNA seed family(miR-21) were perfectly complementary in the seed region with a bulge atpositions 9-12 to prevent RNA interference-type cleavage and degradationof the sponge RNA by Argonaute 2. (Ebert M S, Neilson J R, Sharp P A.Nature Methods. 2007, 4(9):721-726.) Exemplary seed families includelet-7, which has a seed sequence of 5′-GAGGUAG-3′ and comprises Let-7,miR-84, miR-241, and miR-48), and miR30, which has a seed sequence of5′-GUAAACA-3′ and comprises miR30a and miR30e.

Let-7 Sponges

Let-7 represses both the mRNA and proteins levels encoded by Dicer andHMGA2. This repression is not caused by direct cleavage of these twotarget genes; but it may infect the steady-state levels of the targetmRNAs by a mechanism distinct from Argonaute2-catalyzed endonucleolyticcleavage. Because Let-7 regulates expression of the microRNA ProcessingEnzyme, Dicer, it provides a model for studying feedback control by thehuman microRNA.

A rAAV-9-based Let-7 sponge delivery vector was constructed, whichcomprised 5′- and 3′-inverted terminal repeat regions, a CMV immediateearly enhancer, a chicken beta-actin promoter, a luciferase reportergene, one or more sponge sequences (against let-7), and a globin poly-Atail sequence. As a control, an identical vector was a constructedhaving a mut-Let-7 sponge sequence. (FIG. 4, showingrAAV9CB.FFLuc.7XLet7 sponge and rAAV9CB.FFLuc.7X Let7-mutant sponge.)Let7 sponge sequences are shown in FIG. 14. Mice were injected with 10¹²genomic copies of rAAV-9 sponge delivery vector by IV administration.Four weeks after injection luciferase activity was evaluated in the livemice by luciferase imaging. Mice infected with both rAAV9CB.FFLuc.7XLet7sponge and rAAV9CB.FFLuc.7X Let7-mutant sponge exhibited luciferaseactivity throughout. (FIG. 5A.) Luciferase expression was persistent upto four weeks post infection. (FIG. 5B.) Luciferase expression wasconsistently higher in the rAAV9CB.FFLuc.7X Let7-mutant sponge infectedmice compared with rAAV9CB.FFLuc.7XLet7 sponge infected mice, at thesame dose. (FIGS. 6A and B). Copy numbers of let7 and mutant-let7transgenes in cells of the liver were essentially the same between thetwo groups, at the same dose. (FIG. 6C.) This result is consistent withmiR-122 binding specifically to rAAV9CB.FFLuc.7X Let-7 sponge (and notrAAV9CB.FFLuc.7X Let-7-mutant sponge) and inhibiting luciferase proteinexpression.

miR122 Sponges

MiR-122, which is primarily expressed in the liver, inhibits expressionof Cyclin G1, Bcl-w, Cat1 and other genes, promotes HCV replication andregulates lipid and cholesterol metabolism. Elmen et al. have shown thatLNA-antimiR-122 reduces total plasma cholesterol levels in a dosedependent manner (See, e.g., FIGS. 1 and 2 of Elmen J, et al. Nature,2008, 452: 896-900.)

A rAAV-9-based miR-122 sponge delivery vector was constructed, whichcomprised 5′- and 3′-inverted terminal repeat regions, a CMV immediateearly enhancer, a chicken beta-actin promoter, a luciferase reportergene, one or more sponge sequences (against miR-122), and a globinpoly-A tail sequence. As a control, an identical vector was aconstructed having a mutant miR-122 sponge sequence. FIG. 7 shows thestructure of miR122 sponge sequences. C57BL/6 mice were injected with10¹¹ or 10¹² genomic copies of the rAAV-9 sponge delivery vectors by IVadministration. Three weeks after injection luciferase activity wasevaluated in the live mice by luciferase imaging. Mice infected witheither rAAV9TBG.FFLuc.7X miR-122 sponge or rAAV9TBG.FFLuc.7XmiR-122-mutant sponge exhibited luciferase activity mainly in theabdomen. (FIG. 8.) Luciferase expression was dependent on treatment doseand increased in a time-dependent manner, as observed up to three weekspost infection. (FIGS. 9 and 10A.) Luciferase expression wasconsistently higher in the rAAV9TBG.FFLuc.7X miR-122-mutant spongeinfected mice. (FIG. 10B.) This result is consistent with miR-122binding specifically to rAAV9TBG.FFLuc.7X miR-122 sponge (and notrAAV9TBG.FFLuc.7X miR-122-mutant sponge) and inhibiting luciferaseprotein expression. FIG. 13 depicts an miR122 specific rescueexperiment. Mice are infected with LacZ having miR122 binding sites.Expression in the liver is inhibited by endogenous miR122, andinhibition by endogenous miR122 is attenuated when mice are co-infectedwith miR122 sponge.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thisdescription or illustrated in the drawings. The invention is capable ofother embodiments and of being practiced or of being carried out invarious ways. Also, the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having,” “containing,”“involving,” and variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

What is claimed is: 1-20. (canceled)
 21. A method for delivering atransgene to a target tissue in a subject, the method comprisingadministering to the subject a recombinant adeno-associated virus (rAAV)that transduces cells of the target tissue and a transgene, wherein thetransgene is engineered to express an RNA transcript that comprises atleast one binding site for a miRNA that is expressed in cells of anoff-target tissue of the subject.
 22. The method of claim 21, wherein,when the miRNA and the RNA transcript are present together in theoff-target tissue, the miRNA hybridizes to the at least one binding siteof the RNA transcript and inhibits activity of the RNA transcript. 23.The method of claim 21, wherein the miRNA is a liver-specific miRNA andwherein the off-target tissue is liver, or wherein the miRNA is amuscle-specific miRNA and wherein the off-target tissue is muscle. 24.The method of claim 21, wherein the miRNA is miR-122, or miR-1.
 25. Amethod for inhibiting transgene expression in an off-target tissue, themethod comprising administering to a subject a recombinantadeno-associated virus (rAAV) comprising a transgene that is engineeredto express an RNA transcript that comprises at least one binding sitefor a miRNA that is expressed in cells of an off-target tissue of thesubject, wherein, when the miRNA and the RNA transcript are presenttogether in the off-target tissue, the miRNA hybridizes to the at leastone binding site of the RNA transcript and inhibits activity of the RNAtranscript.
 26. The method of claim 25, wherein the transgene isexpressed in a target tissue of the subject.
 27. The method of claim 25,wherein the target tissue is gonad, diaphragm, heart, stomach, liver,spleen, pancreas, muscles, or kidney.
 28. The method of claim 25,wherein the off-target tissue is liver or muscle.
 29. The method ofclaim 25, wherein the RNA transcript is an exogenous miRNA.
 30. Themethod of claim 25, wherein the RNA transcript is an mRNA transcriptthat encodes a protein.
 31. The method of claim 30, wherein the proteinis a therapeutic protein.
 32. The method of claim 25, wherein thetransgene comprises a tissue-specific promoter or an inducible promoteroperatively linked to a coding region from which the RNA transcript isexpressed.
 33. The method of claim 32, wherein the tissue-specificpromoter is a thyroxin binding globulin (TBG) promoter, insulinpromoter, glucagon promoter, somatostatin promoter, pancreaticpolypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase(MCK) promoter, mammalian desmin (DES) promoter, α-myosin heavy chain(α-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.
 34. Themethod of claim 25, wherein the RNA transcript that comprises multiplemiRNA binding sites.
 35. The method of claim 34, wherein each of themultiple miRNA binding sites targets the same miRNA.
 36. The method ofclaim 34, wherein each of the multiple binding sites targets a differentmiRNA.
 37. The method of claim 25, wherein the miRNA is miR-122 ormiR-1.
 38. The method of claim 25, wherein the at least one miRNAbinding site is positioned in the 5′UTR or 3′UTR of the RNA transcript.39. The method claim 25, wherein the rAAV comprises a capsid proteinfrom an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV10, AAV11, or AAV12.
 40. The method of claim 25, whereinthe administration is administration directly to the target tissue.